*Target (protein/gene name): UDP-N-acetylbacillosamine N-acetyltransferase *NCBI Gene # or RefSeq#: 194709137 (Chain A), 194709138 (Chain B), 194709139 (Chain C), 123141428 (Full) *Protein ID (NP or XP #) or Wolbachia#: N/A *Organism (including strain): Campylobacter jejuni Etiologic Risk Group (see link below): 2 http://www.absa.org/riskgroups/bacteriasearch.php?genus=Campylobacter&species=jejuni
*Background/Disease Information (sort of like the Intro to your Mini Research Write up):
Campylobacteriosis is a bowel disease caused by the bacteria Campylobacter jejuni. This disease is also known as campylobacter enteritis and gastroenteritis. C.jejuni is most commonly found in animal feces, and it infects the humans through accidental consumption of unclean food. This type of bacteria requires reduced levels of oxygen (3-5% oxygen and 2-10% carbon dioxide levels) in order to grow. In animals, C.jejuni is known to cause abortion and enteritis, specifically in sheep and cattle. Humans develop food poisoning, diarrhea, enteritis, and a case as severe as Crohn's disease in rare instances. Campylobacter jejuni is the leading bacteria that causes diarrheal illness in the United States.
Essentiality of this protein:
"This type of structure is found in many enzymes that employ CoA-linked donors, including a sugar O-acetyltransferase fromEscherichia coli, GAT, which modifies some galactose species to prevent them triggering the lac operon." http://www.ncbi.nlm.nih.gov/pubmed/18198901
"We show that Cj1123c has acetyl-CoA-dependent N-acetyltransferase activity not only on the UDP-4-amino-4,6-dideoxy-GlcNAc intermediate of the N-glycosylation pathway but also on the UDP-4-amino-4,6-dideoxy-AltNAc intermediate of the O-glycosylation pathway, implying functional redundancy between both pathways. We further demonstrate that, despite its somewhat relaxed substrate specificity for N-acetylation, Cj1123c cannot acetylate aminoglycosides, indicating a preference for sugar-nucleotide substrates." http://www.ncbi.nlm.nih.gov/pubmed/19448740
Complex of proteins?: 3 Chains. Druggable Target (list number or cite evidence from a paper/database showing druggable in another organism): N/A
Figure 1:Reaction schematic of UDP-N-acetylbacillosamine N-acetyltransferase with acetyl CoA from BRENDA.
Enzyme Assay information (spectrophotometric, coupled assay ?, reagents): N/A -- link to Sigma (or other company) page for assay (see Sigma links below) -- -or link (or citation) to paper that contains assay information -- links to assay reagents (substrates) pages. --- List cost and quantity of substrate reagents, supplier, and catalog #
Structure Available (PDB or Homology model) PDB #: 3BSY
Current Inhibitors: NONE
Expression Information (has it been expressed in bacterial cells):
It's been expressed in Escherichia coli. Figure 2: Screenshot of expression information for 3BSY in BRENDA.
Purification Method:
"The UDP-N,N‘-diacetylbacillosamine product was purified from the reaction by reverse phase C18 HPLC and the structure determined by NMR analysis. Additionally, the full-length PglF was overexpressed and purified in the presence of detergent as a GST fusion protein, allowing for derivation of kinetic parameters (Olivier)."
Image of protein (PyMol with features delineated and shown separately):
Figure 3: Cartoon image of protein 3BSY with the 3 chains shown in 3 different colors.
Figure 4: The protein 3BSY shown in sticks. The 3 chains are colored by element with carbon in 3 different colors: carbon in green, blue, then pink. The red represents oxygen, while the blue is for nitrogen.
*Amino Acid Sequence (paste as text only - not as screenshot or as 'code'): MARTEKIYIYGASGHGLVCEDVAKNMGYKECIFLDDFKGMKFESTLPKYDFFIAIGNNEIRKKIYQKISE NGFKIVNLIHKSALISPSAIVEENAGILIMPYVVINAKAKIEKGVILNTSSVIEHECVIGEFSHVSVGAK CAGNVKIGKNCFLGINSCVLPNLSLADDSILGGGATLVKNQDEKGVFVGVPAKRM
*length of your protein in Amino Acids: 198 amino acids (for 1 chain)
Molecular Weight of your protein in kiloDaltons using the Expasy ProtParam website: 21.147 kDA
Molar Extinction coefficient of your protein at 280 nm wavelength:
Figure 5: Screenshot of the Extinction coefficients of 3BSY from Expasy ProtParam.
*CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only):Can't be found. *GC% Content for gene (codon optimized):Can't be found.
Do Not Need this info for Spring (but still copy these lines to your Target page for now) Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers): (link to DNA Works output text file - that should be saved in your Google Docs folder after you did the primer design protocol) -- Ask a mentor, Dr. B, or a fellow researcher -how to link a GDocs file if you are not sure how to.
Primer design results for 'tail' primers (this is just 2 sequences):
*NCBI Gene # or RefSeq#: 194709137 (Chain A), 194709138 (Chain B), 194709139 (Chain C), 123141428 (Full)
*Protein ID (NP or XP #) or Wolbachia#: N/A
*Organism (including strain): Campylobacter jejuni
Etiologic Risk Group (see link below): 2
http://www.absa.org/riskgroups/bacteriasearch.php?genus=Campylobacter&species=jejuni
*Background/Disease Information (sort of like the Intro to your Mini Research Write up):
Campylobacteriosis is a bowel disease caused by the bacteria Campylobacter jejuni. This disease is also known as campylobacter enteritis and gastroenteritis. C. jejuni is most commonly found in animal feces, and it infects the humans through accidental consumption of unclean food. This type of bacteria requires reduced levels of oxygen (3-5% oxygen and 2-10% carbon dioxide levels) in order to grow. In animals, C. jejuni is known to cause abortion and enteritis, specifically in sheep and cattle. Humans develop food poisoning, diarrhea, enteritis, and a case as severe as Crohn's disease in rare instances. Campylobacter jejuni is the leading bacteria that causes diarrheal illness in the United States.
Link to TDR Targets page (if present): N/A
Link to Gene Database page (NCBI, EuPath databases -e.g. TryTryp, PlasmoDB, etc - or PATRIC, etc.):
Chain A: http://www.ncbi.nlm.nih.gov/protein/3BSY_A
Chain B: http://www.ncbi.nlm.nih.gov/protein/3BSY_B
Chain C: http://www.ncbi.nlm.nih.gov/protein/3BSY_C
Full: http://www.ncbi.nlm.nih.gov/protein/Q0P9D1.1
Essentiality of this protein:
"This type of structure is found in many enzymes that employ CoA-linked donors, including a sugar O-acetyltransferase fromEscherichia coli, GAT, which modifies some galactose species to prevent them triggering the lac operon."
http://www.ncbi.nlm.nih.gov/pubmed/18198901
"We show that Cj1123c has acetyl-CoA-dependent N-acetyltransferase activity not only on the UDP-4-amino-4,6-dideoxy-GlcNAc intermediate of the N-glycosylation pathway but also on the UDP-4-amino-4,6-dideoxy-AltNAc intermediate of the O-glycosylation pathway, implying functional redundancy between both pathways. We further demonstrate that, despite its somewhat relaxed substrate specificity for N-acetylation, Cj1123c cannot acetylate aminoglycosides, indicating a preference for sugar-nucleotide substrates."
http://www.ncbi.nlm.nih.gov/pubmed/19448740
Complex of proteins?: 3 Chains.
Druggable Target (list number or cite evidence from a paper/database showing druggable in another organism): N/A
*EC#: 2.3.1.203
Link to BRENDA EC# page: http://www.brenda-enzymes.info/php/result_flat.php4?ecno=2.3.1.203
Figure 1: Reaction schematic of UDP-N-acetylbacillosamine N-acetyltransferase with acetyl CoA from BRENDA.
Enzyme Assay information (spectrophotometric, coupled assay ?, reagents): N/A
-- link to Sigma (or other company) page for assay (see Sigma links below)
-- -or link (or citation) to paper that contains assay information
-- links to assay reagents (substrates) pages.
--- List cost and quantity of substrate reagents, supplier, and catalog #
Structure Available (PDB or Homology model)
PDB #: 3BSY
Current Inhibitors: NONE
Expression Information (has it been expressed in bacterial cells):
It's been expressed in Escherichia coli.
Figure 2: Screenshot of expression information for 3BSY in BRENDA.
Purification Method:
"The UDP-N,N‘-diacetylbacillosamine product was purified from the reaction by reverse phase C18 HPLC and the structure determined by NMR analysis. Additionally, the full-length PglF was overexpressed and purified in the presence of detergent as a GST fusion protein, allowing for derivation of kinetic parameters (Olivier)."
http://pubs.acs.org.ezproxy.lib.utexas.edu/doi/full/10.1021/bi061456h
Image of protein (PyMol with features delineated and shown separately):
Figure 3: Cartoon image of protein 3BSY with the 3 chains shown in 3 different colors.
Figure 4: The protein 3BSY shown in sticks. The 3 chains are colored by element with carbon in 3 different colors: carbon in green, blue, then pink. The red represents oxygen, while the blue is for nitrogen.
*Amino Acid Sequence (paste as text only - not as screenshot or as 'code'):
MARTEKIYIYGASGHGLVCEDVAKNMGYKECIFLDDFKGMKFESTLPKYDFFIAIGNNEIRKKIYQKISE
NGFKIVNLIHKSALISPSAIVEENAGILIMPYVVINAKAKIEKGVILNTSSVIEHECVIGEFSHVSVGAK
CAGNVKIGKNCFLGINSCVLPNLSLADDSILGGGATLVKNQDEKGVFVGVPAKRM
*length of your protein in Amino Acids: 198 amino acids (for 1 chain)
Molecular Weight of your protein in kiloDaltons using the Expasy ProtParam website: 21.147 kDA
Molar Extinction coefficient of your protein at 280 nm wavelength:
Figure 5: Screenshot of the Extinction coefficients of 3BSY from Expasy ProtParam.
TMpred graph Image (http://www.ch.embnet.org/software/TMPRED_form.html). Input your amino acid sequence to it.
Figure 6: TMPred output graph for C. jejuni protein 3BSY.
*CDS Gene Sequence (paste as text only):
atggcgcgcaccgaaaaaatttatatttatggcgcgagcggccatggcctggtgtgcgaa gatgtggcgaaaaacatgggctataaagaatgcatttttctggatgattttaaaggcatg aaatttgaaagcaccctgccgaaatatgatttttttattgcgattggcaacaacgaaatt cgcaaaaaaatttatcagaaaattagcgaaaacggctttaaaattgtgaacctgattcat aaaagcgcgctgattagcccgagcgcgattgtggaagaaaacgcgggcattctgattatg ccgtatgtggtgattaacgcgaaagcgaaaattgaaaaaggcgtgattctgaacaccagc agcgtgattgaacatgaatgcgtgattggcgaatttagccatgtgagcgtgggcgcgaaa tgcgcgggcaacgtgaaaattggcaaaaactgctttctgggcattaacagctgcgtgctg ccgaacctgagcctggcggatgatagcattctgggcggcggcgcgaccctggtgaaaaac caggatgaaaaaggcgtgtttgtgggcgtgccggcgaaacgcatg
*GC% Content for gene: 30.83%
http://www.kazusa.or.jp/codon/cgi-bin/showcodon.cgi?species=192222
*CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only): Can't be found.
*GC% Content for gene (codon optimized): Can't be found.
Do Not Need this info for Spring (but still copy these lines to your Target page for now)
Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers):
(link to DNA Works output text file - that should be saved in your Google Docs folder after you did the primer design protocol)
-- Ask a mentor, Dr. B, or a fellow researcher -how to link a GDocs file if you are not sure how to.
Primer design results for 'tail' primers (this is just 2 sequences):